Dermatological compositions and methods

ABSTRACT

Disclosed are methods and compositions for regulating the melanin content of mammalian melanocytes; regulating pigmentation in mammalian skin, hair, wool or fur; treating or preventing various skin and proliferative disorders; by administration of various compounds, including alcohols, diols and/or triols and their analogues.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of PCT/US98/05346filed Mar. 18, 1998, which is a continuation-in-part of PCT/US97/16642filed Sep. 18, 1997, which is a continuation-in-part of application Ser.No. 08/933,143 filed Sep. 18, 1997, which is a continuation-in-part ofapplication Serial No. 60/026,577 filed Sep. 18, 1996, of applicationSerial No. 60/035,947 filed Jan. 21, 1997, of application Serial No.60/036,863 filed Feb. 4, 1997, and of application Serial No. 60/048,597filed Jun. 4, 1997.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not Applicable

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates to regulating the melanin contentof mammalian melanocytes; regulating pigmentation in mammalian skin,hair, wool or fur; restoring pigmentation to grey hair; treating orpreventing various skin and proliferative disorders; by administrationof various compounds, including alcohols, diols and/or triols and theiranalogues.

[0005] 2. Description of Related Art

[0006] U.S. Pat. No. 5,352,440 is directed to increasing melaninsynthesis in melanocytes and increasing pigmentation by administrationof certain diacylglycerol compounds.

[0007] U.S. Pat. No. 5,532,001 is directed to increasing pigmentation inmammalian skin via administration of certain DNA fragments.

[0008] U.S. Pat. No. 5,554,359 is directed to increasing levels ofmelanin in melanocytes by administration of lysosomotropic agents.

SUMMARY OF THE INVENTION

[0009] The present invention provides a method for increasing themelanin content of mammalian melanocytes, which comprises administeringto said melanocytes an effective amount of a C₃-C₅₀ diol, which may bealiphatic or aromatic, linear, branched, mono-, bi- or polyclicic,saturated or unsaturated, unsubstituted, mono- or polysubstituted.

[0010] Another aspect of the present invention concerns a method forincreasing or restoring pigmentation in mammalian skin, hair or wool,which comprises administering to said mammal an effective amount of oneor more compounds described above.

[0011] Another aspect of the present invention concerns a method fortreating a skin proliferative disorder or a disorder of keratinizationin a mammal, which comprises administering to a mammal in need of suchtreatment an effective amount of one or more compounds described above.

[0012] A further aspect of the present invention concerns a method forpreventing a skin proliferative disorder or a disorder of keratinizationin a mammal, which comprises administering to a mammal in need of suchpreventive treatment an effective amount of one or more compoundsdescribed above.

[0013] An additional aspect of the present invention concerns a methodfor treating a tumorous or cancerous disorder whereby application of oneor more of the compounds described above results in reversal of saiddisorder by virtue of induction of differentiation of cancerous ortumorous cells to a less- or non-proliferative phenotype. Thesecancerous or tumorous disorders include, but are not limited to,proliferative disorders of a dermatological nature.

[0014] In another aspect, the present invention provides a compositionfor increasing the melanin content of mammalian melanocytes, whichcomprises:

[0015] a) an effective amount of one or more compounds described above;and

[0016] b) a suitable carrier.

[0017] In another aspect, the present invention provides a compositionfor treating a skin proliferative disorder or a disorder ofkeratinization, which comprises:

[0018] a) an effective amount of one or more compounds described above;and

[0019] b) a suitable carrier.

[0020] In yet another aspect, the present invention provides acomposition for preventing a skin proliferative disorder, whichcomprises:

[0021] a) an effective amount of one or more compounds described above;and

[0022] b) a suitable carrier.

[0023] The present invention additionally provides a method forincreasing the melanin content of mammalian melanocytes, which comprisesadministering to said melanocytes an effective amount of one or morecompounds having the following structure:

[0024] wherein

[0025] each X is independently selected from a single or double bond; ora group containing from one atom to twenty atoms, at least one of whichis carbon, nitrogen, oxygen or sulfur;

[0026] each R₁ is independently selected from hydrogen; halogen; an acylor amino acyl group containing from one atom to twenty atoms, at leastone of which is carbon, nitrogen, oxygen, or sulfur; or a groupcontaining from one atom to twenty atoms, one of which is carbon,nitrogen, oxygen, or sulfur;

[0027] R₂ is a linear, branched or unbranched, cyclic, bicyclic orpolycyclic group containing from one atom to fifty atoms, at least oneof which is carbon, nitrogen, oxygen, or sulfur; and

[0028] each R is independently selected from R₁; R₂; hydroxyl, methyl,hydroxymethyl, —(CH₂)_(n)CH₃—, —(CH₂)_(n)OH, —(CH₂)_(n)OR₁,—(CH₂)_(n)—CH(OH)—CHOH, —(CH₂)_(n)—CH(OH)—CH(OH)R₁,—(CH₂)_(n)—CH(OH)—(CH₂)_(n)—CH₂(OH),—(CH₂)_(n)—CH(OH)—(CH₂)_(n)—CH(OH)R₁ or —CH₂OR₁, wherein each n isindependently an integer from 0-25;

[0029] and pharmaceutically acceptable salts or prodrugs thereof, withthe proviso that with reference to the first listed structure only, whenthe X to which R₁ is attached is a single bond and each R is acyl andone of R₁ is hydroxymethyl (HOCH₂—), then the sum of carbon atoms in R₁is greater than one.

[0030] Another aspect of the present invention concerns a method forincreasing or restoring pigmentation in mammalian skin, hair or wool,which comprises administering to said mammal an effective amount of oneor more compounds depicted above.

[0031] Another aspect of the present invention concerns a method fortreating a skin proliferative disorder or a disorder of keratinizationin a mammal, which comprises administering to a mammal in need of suchtreatment an effective amount of one or more compounds depicted above.

[0032] A further aspect of the present invention concerns a method forpreventing a skin proliferative disorder or a disorder of keratinizationin a mammal, which comprises administering to a mammal in need of suchpreventive treatment an effective amount of one or more compoundsdepicted above.

[0033] An additional aspect of the present invention concerns a methodfor treating a tumorous or cancerous disorder whereby application of oneor more of the compounds depicted above results in reversal of saiddisorder by virtue of induction of differentiation of cancerous ortumorous cells to a less- or non-proliferative phenotype. Thesecancerous or tumorous disorders include, but are not limited to,proliferative disorders of a dermatological nature.

[0034] In another aspect, the present invention provides a compositionfor increasing the melanin content of mammalian melanocytes, whichcomprises:

[0035] a) an effective amount of one or more compounds depicted above;and

[0036] b) a suitable carrier.

[0037] In another aspect, the present invention provides a compositionfor treating a skin proliferative disorder or a disorder ofkeratinization, which comprises:

[0038] a) an effective amount of one or more compounds depicted above;and

[0039] b) a suitable carrier.

[0040] In yet another aspect, the present invention provides acomposition for preventing a skin proliferative disorder, whichcomprises:

[0041] a) an effective amount of one or more compounds depicted above;and

[0042] b) a suitable carrier.

[0043] In yet another aspect, the present invention provides a method ofaltering or restoring pigmentation in mammalian skin, hair, wool or fur,which comprises administering to a mammal an effective amount of acompound which alters cellular production of nitric oxide, wherein anincrease in nitric oxide production results in increased pigmentation,and a decrease in nitric oxide production results in decreasedpigmentation.

[0044] In yet another aspect, the present invention provides a method ofaltering pigmentation in mammalian skin, hair, wool or fur, whichcomprises administering to a mammal an effective amount of a compoundwhich alters cellular production of cyclic guanosine monophosphate,wherein an increase in cyclic guanosine monophosphate production resultsin increased pigmentation, and a decrease in cyclic guanosinemonophosphate production results in decreased pigmentation.

[0045] In yet another aspect, the present invention provides a method ofaltering pigmentation in mammalian skin, hair, wool or fur, whichcomprises administering to a mammal an effective amount of a compoundwhich alters cellular activity of protein kinase G, wherein an increasein protein kinase G activity results in increased pigmentation, and adecrease in protein kinase G activity results in decreased pigmentation.

[0046] In yet another aspect, the present invention provides a method ofidentifying a substance which alters pigmentation in mammalianmelanocytes, which comprises evaluating the effect the substance has oncellular production of nitric oxide, wherein if such production isaltered, then the pigmentation in mammalian melanocytes is altered.

[0047] In yet another aspect, the present invention provides a method ofidentifying a substance which alters pigmentation in mammalianmelanocytes, which comprises evaluating the effect the substance has oncellular production of cyclic guanosine monophosphate, wherein if suchproduction is altered, then the pigmentation in mammalian epidermalmelanocytes is altered.

[0048] In yet another aspect, the present invention provides a method ofidentifying a substance which alters pigmentation in mammalianmelanocytes, which comprises evaluating the effect the substance has oncellular activity of protein kinase G, wherein if such activity isaltered, then the pigmentation in mammalian epidermal melanocytes isaltered.

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] FIGS. 1A-1D are printouts from an Oncor Imaging Systems ofFontana-Masson stained guinea pig skin biopsy samples as described inExample 5.

[0050]FIG. 2 is a series of bar graphs depicting the structure activityresults obtained in Example 7.

[0051] FIGS. 3A-3D are printouts of normal human epidermal melanocytesand melanosomes as described in Example 8.

[0052]FIG. 4 is a series of bar graphs depicting the structure activityresults obtained in Example 12.

[0053] FIGS. 5A-5B are photographs of treated guinea pig skin asdescribed in Example 13.

[0054] FIGS. 6A-6D are printouts as described in Example 13.

DETAILED DESCRIPTION OF THE INVENTION

[0055] The present invention is based on the unique observation thatcertain compounds effectively and efficiently induce melanogenesis inmammalian cells, which has several consequences. First, increasingmelanogenesis leads to increasing the melanin content of melanocytes,and hence results in increased pigmentation or darkened color of theskin, hair wool or fur. Thus, the present invention is useful in thetreatment of hypopigmentation disorders, such as albinism, vitiligo,etc. It is also believed that increasing the pigmentation of skinaccording to the present invention will protect such skin fromsubsequent UV light damage, sunburn, photoaging and development of skincancers. Finally, since the methods and compositions described hereininduce differentiation of a melanoma cell line, the present inventionmay be used to treat hyperproliferative disorders such as actinickeratosis, basal cell carcinoma, squamous cell carcinoma, fibroushistiocytoma, dermatofibrosarcoma protuberans, hemangioma, nevusflammeus, xanothoma, Kaposi's sarcoma, mastocytosis, mycosis fungoides,lentigo, nevocellular nevus, lentigo maligna, malignant melanoma, andmetastatic carcinoma.

[0056] The present methods and compositions are also useful in thetreatment of diseases characterized by inflammation and disturbance ofkeratinization, including psoriasis vulgaris, psoriasis eosinophilia,acne vulgaris, acne conglobata, acne fulminans, osteoma cutis,nodulocystic acne, cystic acne and benign and premalignant dermatoses.

[0057] The active compounds according to the present invention are theC₃-C₅₀ diols described above (by “diol” is meant a compound which has atleast two, but permissibly more, —OH groups). Preferably, the activehave one of the six structures depicted above. More preferably, X isindependently selected from a single bond; or C₁-C₁₀ alkylene, C₂-C₁₀alkenylene, or C₂-C₁₀ alkynylene, each of which may contain one or moredifferent heteroatoms or heteroatoms of the same type. More preferablyeach R₁ is independently selected from hydrogen; fluoro; chloro; orC₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, C₇-C₂₀ aralkyl, C₈-C₂₀aralkenyl, C₈-C₂₀ aralkinyl, or C₆-C₂₀ aryl, each of which may containone or more different heteroatoms or heteroatoms of the same type, orcarboxyl, carboxamido, carbalkoxy, sulfamido, sulfonamido; hydroxyl, oramino. More preferably R₂ contains from two to twenty carbon atoms, eachmay contain one or more different heteroatoms or heteroatoms of the sametype.

[0058] The preparation of the present compounds would be apparent to oneof ordinary skill, and many of them are commercially available.Representative preferred compounds include, but are not limited to:

[0059] 1,2-Ethanediol

[0060] 1,2-Propanediol(Propylene Glycol)

[0061] (S)-(+)-1,2-Propanediol[(S)-(+)-1,2-Propylene Glycol]

[0062] 1,3-Propanediol

[0063] 2,3-Dimethyl-2,3-Butanediol

[0064] 2,3-Dimethyl-1,2-Butanediol

[0065] 1-Phenyl-1,2-Propanediol

[0066] 2-Methyl-1,3-Propanediol

[0067] 1,2-Butanediol

[0068] 1,3-Butanediol

[0069] 1,4-Butanediol

[0070] 2,3-Butanediol

[0071] (2R,3R)-(−)-2,3-Butanediol

[0072] (2S,3S)-(+)-2,3-Butanediol

[0073] 2,3-meso-Butanediol

[0074] 1,2-Pentanediol

[0075] 1,4-Pentanediol

[0076] 1,5-Pentanediol

[0077] 2,4-Pentanediol

[0078] 1,2-cis-cyclopentanediol

[0079] 1,2-trans-cyclopentanediol

[0080] 1,2-cis-cyclohexaneanediol

[0081] 1,2-trans-cyclohexanediol

[0082] 1,2-dihydroxy-4,5-cyclohexanediol carbonate

[0083] 1,2,4,5-tetrahydroxycyclohexane

[0084] 1,2-Hexanediol

[0085] 1,5-Hexanediol

[0086] 1,6-Hexanediol

[0087] 2,5-Hexanediol

[0088] 1,2-Heptanediol

[0089] 1,7-Heptanediol

[0090] 7-Octene-1,2-diol

[0091] 1,2-Octanediol

[0092] 1,8-Octanediol

[0093] 1,2-Nonanediol

[0094] 1,9-Nonanediol

[0095] 1,2-Decanediol

[0096] 1,10-Decanediol

[0097] 1,2-Dodecanediol

[0098] 1,12-Dodecanediol

[0099] 1,2-Tetradecanediol

[0100] 1,14-Tetradecanediol

[0101] 1,2-Hexadecanediol

[0102] 1,16-Hexadecanediol

[0103] Glycerol

[0104] 1,2,4-Butanetriol

[0105] 1,2,3-Trihydroxyhexane

[0106] 1,2,6-Trihydroxyhexane

[0107] 1,2,3-Heptanetriol

[0108] β-estradiol

[0109] azabicyclo-(2,2,1)-heptanediol-3-one

[0110] 1,4-dioxane-2,3-diol

[0111] 5-norbornene-2,2-dimethanol

[0112] norbornane-2,2-dimethanol

[0113] 2,3-norbornanediol (exo or endo or cis or trans)

[0114] 2,3-cis-exo-norbornanediol

[0115] α-norborneol

[0116] 2-norbornanemethanol

[0117] norbornane

[0118] borneol

[0119] camphor

[0120] camphene

[0121] camphane

[0122] norbornane acetic acid

[0123] norbornane-carboxylic acid

[0124] norbornane-dicarboxylic acid

[0125] 2-endo-hexadecylamino-5-norbornene-2-exo-methanol

[0126] 2-endo-hexadecylamino-5-norbornene-2,3-exo-dimethanol

[0127] 2-(propyl-1,2-diol)-norbornane

[0128] 1,2-dithiane-trans-4,5-diol

[0129] 2,3-pyridinediol

[0130] 2,3-pyridinediol hydrogen chloride

[0131] 2,3-pyridinediol glycolic acid

[0132] 2,3-dipyridyl-2,3-butanediol

[0133] 2,2,4,4-tetramethyl-1,3-cyclobutanediol

[0134] norborneol

[0135] 2,7-norbornanediol

[0136] 2,5,7-norbornanetriol

[0137] 2,6,7-norbornanetriol

[0138] 2-hydroxy-2-norbornanemethanol

[0139] 1-(exo-2-norbornyl-)-propan-1,2-diol

[0140] 1-(endo-2-norbornyl-)-propan-1,2-diol

[0141] methyl-5-norbornene-2,3-dimethanol

[0142] 2-norbornaneacetic acid

[0143] 1,2-cis-cyclohexanedimethanol

[0144] 3-cyclohexane-1,1-dimethanol

[0145] 1,4-cyclohexanedimethanol

[0146] pentaerylthritol

[0147] pinane

[0148] pinaneol

[0149] 2,3-cis/exo-pinanediol([1R,2R,3S,5R]-[−]-pinanediol and[1S,2S,3R,5S]-[+)-pinanediol])

[0150] (1R)-(−)-trans-pinane-1,10-diol

[0151] (1S,2S,5S,)-2-hydroxy-3-pinanone

[0152] (−)-isopinocampheol

[0153] (S)-cis-verbenol

[0154] bornane

[0155] borneol

[0156] 2,3-cis/exo-bornanediol

[0157] 2,3-trans-bornanediol

[0158] camphanediol

[0159] camphenediol

[0160] cis-p-menthane-3,8-diol

[0161] trans-p-menthane-3,8-diol

[0162] sobrerol(trans-p-meth-6-ene-2,8-diol)

[0163] α-terpineol

[0164] terpinen-4-ol

[0165] (−)-cis-myrtanol[(1S,2R)-10-Pinanol]

[0166] (+)-trans-myrtanol[(1R,2R)-10-Pinanol]

[0167] (−)-trans-myrtanol[(1S,2S)-10-Pinanol]

[0168] (−)-myrtenal[(1R)-2-Pinen-10-al]

[0169] (−)-myrtenol[(1R)-2-Pinene-10-ol]

[0170] carveol[p-mentha-6,8-dien-2-one]

[0171] menthol

[0172] Particularly preferred compounds of this invention are2,3-cis/exo-pinanediol([1R,2R,3S,5R]-[−]-pinanediol and[1S,2S,3R,5S]-[+]-pinanediol]; 2,3-cis/exo-bornanediol;5-norbornene-2,2-dimethanol; norbornane-2,2-dimethanol;2-hydroxy-2-norbornanemethanol; 1-(exo-2-norbornyl-)-propan-1,2-diol;and 1-(endo-2-norbornyl-)-propan-1,2-diol. Other preferred compounds are(1S,2S,5S,)-2-hydroxy-3-pinanone; 2,3-trans-pinanediol;(1R)-(−)-trans-pinane-1,10-diol; 2,3-trans-bornanediol;cis-p-menthane-3,8-diol; trans-p-menthane-3,8-diol;1,2-cis-cyclopentanediol, 2,3-cis/exo-norbornanediol;2-norbornanemethanol; (1R)-(−)-myrtenol, and3,3-dimethyl-1,2-butanediol.

[0173] The methods and compositions of the present invention contemplatethe use of one or more of the above-mentioned compounds as an activeingredient for various uses. In a preferred embodiment, the activeingredient(s) is combined with an acceptable carrier to form a topicalformulation which may be placed on the skin for dermatological uses.Topical formulations may include ointments, lotions, pastes, creams,gels, drops, suppositories, sprays, liquids, shampoos, powders andtransdermal patches. Thickeners, diluents, emulsifiers, dispersing aidsor binders may be used as needed. Preferably, one function of thecarrier is to enhance skin penetration of the active ingredient(s), andshould be capable of delivering the active ingredient(s) to melanocytesunder in vivo conditions. Suitable carriers are well known to one ofordinary skill, and include liposomes, ethanol, dimethylsulfoxide(DMSO), petroleum jelly (petrolatum), mineral oil (liquid petrolatum),water, dimethylformamide, dekaoxyethylene-oleylether, oleic acid,2-pyrrolidone and Azone® brand penetration enhancer (Upjohn). Aparticularly preferred composition includes an active ingredient(s) asdescribed above, with one of 2-pyrrolidone, oleic acid and/or Azone® aspenetration enhancer, solubilized in a base of water, ethanol, propanoland/or propylene glycol (the latter component having properties of acarrier, penetration enhancer and an active ingredient as describedherein). Depending on the specific application, the compositions of thepresent invention may also include other active ingredients, as well asinert or inactive ingredients.

[0174] Particularly preferred formulations include an activeingredient(s) in conjunction with one or more melanogenesis-enhancingagents such as α-hydroxy acids, salts and derivatives thereof; α-ketoacids, salts and derivatives thereof; β-hydroxy acids, salts andderivatives thereof; retinoids, salts and derivatives thereof; Vitamin Aand related compounds; acids; phenol; and methoxypropyl-gluconamide, asmore fully described in co-pending application Ser. No. ______ filedApr. 6, 1998 entitled “Dermatological Formulations and Methods”, thecontents of which are incorporated herein by reference.

[0175] The dose regimen will depend on a number of factors which mayreadily be determined, such as severity and responsiveness of thecondition to be treated, but will normally be one or more doses per day,with a course of treatment lasting from several days to several months,or until a cure is effected or a diminution of disease state isachieved, or a cosmetically desired degree of melanogenesis (tanning) isachieved, depending on the application. One of ordinary skill mayreadily determine optimum dosages, dosing methodologies and repetitionrates. In general, it is contemplated that topical formulations (such ascreams, lotions, solutions, etc.) will have a concentration of activeingredient of from about 0.01% to about 50%, preferably from about 0.1%to about 10%. In general, it is contemplated that unit dosage formcompositions according to the present invention will contain from about0.01 mg to about 100 mg of active ingredient, preferably about 0.1 mg toabout 10 mg of active ingredient.

[0176] Another aspect of the present invention is based on theobservation that the subject compounds which stimulate melaninproduction act via the Nitric Oxide/cyclic Guanosinemonophosphate/Protein Kinase G (“NO/cGMP/PKG”) pathway. Thus, thepresent invention includes not only the compounds described above, butany compound which acts via the NO/cGMP/PKG pathway to stimulate melaninsynthesis by increasing cellular production of NO, cGMP or PKG.Conversely, agents which decrease cellular production of NO, cGMP or PKGwill decrease or suppress melanin production and pigmentation inmammalian skin, hair, fur or wool, and the present invention is alsodirected to those compositions and methods. Such is useful in, forexample, the lightening of skin, hair, wool or fur for cosmeticpurposes, or the treatment of hyperpigmentation or uneven pigmentationdisorders such as vitiligo, dermal melanocytosis,Franceschetti-Jadassohn Syndrome, etc. For such depigmentationapplications, the formulation and dosing would be as described abovewith respect to pigmentation applications.

[0177] Discovery of the pathway through which the present compounds actalso leads to methods for screening compounds for melanogenic activityand potency, or for their ability to reduce or suppress melanogenesis,based on measurement of generation of nitric oxide (NO) or measurementof nitric oxide synthesis (NOS) activity. Methods for measurement of NOor NOS include but are not limited to the following well known methods.Measurement of NO is usually based on the fact that NO rapidlydecomposes to nitrate and nitrite in aqueous solution. Nitrate reductaseis added to culture media or cell extracts to ensure complete conversionof nitrate to nitrite. Griess reagents (sulfanilamide andN-[1-naphthyl]-ethylenediamine) are then added to convert nitrite into adeep purple azo compound that absorbs maximally at 540 nm (Schmidt, etal., 1995, Biochemica 2:22). Reactions are typically carried out in a96-well format with absorbances read on a microtiter plate reader.Alternatively, following conversion of nitrate to nitrite as describedabove, DAN reagent (2,3-diaminonaphthalene) is added followed by NaOHwhich converts nitrite into the fluorescent compound1(H)-naphthotriazole. This is measured fluorimetrically with excitationat 365 nm and emission at 450 nm, typically in a 96-well format (Miles,et al., 1995, Methods 7:40). NOS activity is measured by adding[³H]-arginine to intact tissues or protein extracts, and measuringrelease of ³H resulting from the conversion of arginine to citrullineduring the enzymatic formation of NO by NOS (Baudouin and Tachon, 1996,J. Invest. Dermatol. 106:428-431). Alternatively, the production of cGMPor activity of PKG can be used as a screening tool. cGMP may be measuredby commercially available immunoassay (see Romero-Graillet, et al.,1996, J. Biol. Chem. 271:28052-28056). PKG may be measured by cyclic GMPdependent kination of a primary histone target (see Hidaka, et al.,Biochemistry 1984, 23, 5036-5041)

[0178] The use of and useful and novel features of the present methodsand compositions will be further understood in view of the followingnon-limiting examples.

EXAMPLE 1

[0179] The Cloudman S91 mouse melanoma cell line was obtained fromAmerican Type Culture Collection (ATCC). Cells were cultured inDulbecco's Modified Eagles Medium (DMEM) containing 10% calf serum, 2 mML-glutamine, 10 U Penicillin/ml and 10 ug Streptomycin/ml according to apreviously published protocol (Eller, et al., Proc. Natl. Acad. Sci.93:1087-92. 1996). For testing propylene glycol and analogues forinduction of melanogenesis, S91 cells were plated at 10⁵ cells/35 mmdish in 10% calf serum. One day after plating, media was removed andreplaced with media containing 2% calf serum and test compounds (Eller,et al., 1996). Cells were cultured for 6 days at 37° C. in 5% CO₂ in ahumidified incubator. Following this treatment period, cells wereexamined microscopically and the portion of dedifferentiated anddifferentiated cells was estimated. Previous studies have shown thatdedifferentiated S91 cells have a rounded, spindly appearance whiledifferentiated S91 cells have a flattened, cuboidal, multipolar anddendritic appearance (Orlow, et al., Exp. Cell Res. 191:209-218, 1990).

[0180] Following this microscopic examination, cells were detached fromdishes by trypsin. The time required for detachment by trypsin wasrecorded as an additional indicator of the phenotypic effects of testcompounds. For each treatment, a subsample of cells was counted todetermine the effects of treatment compounds on cellular proliferation.The remainder of cells were used for determination of melanin content.Melanin was extracted from cells by vortexing for 15 min in 1N NaOH.Standards were prepared by dissolving melanin (Sigma) in 1 N NaOH(Eller, et al., 1996). Absorbance of standards and samples was measuredat 475 nm. Melanin was expressed as pg melanin/cell.

[0181] Tables 1 and 2 below show the results obtained when testingformulations containing various concentrations of 1,2-propanediol as theactive ingredient. In the control, no test compound was added to themedium. TABLE 1 Concentration Cells (× 10⁶) ug Melanin pg Melanin/CellControl 0.48 2.52 5.3 1% (136 mM) 0.52 4.88 9.4 2% (272 mM) 0.50 6.2412.5 3% (408 mM) 0.20 4.03 20.2 4% (544 mM) 0.10 4.01 40.1 5% (680 mM)0.08 2.31 28.9

[0182] TABLE 2 Morphology Rounded Flattened Trypsinization ConcentrationSpindly Cuboidal Detachment Time Control 100%  ≦3 min 1% (136 mM)  90% 10%  ≦6 min 2% (272 mM)  70%  30%  ≦9 min 3% (408 mM)  40%  60% ≦12 min4% (544 mM)  15%  85% ≦15 min 5% (680 mM) 100% ≦15 min

EXAMPLE 2

[0183] The same procedure as in Example 1 was followed, except thatethanol, and isomers of propanediol and butanediol were used as testcompounds. The results are set forth in Tables 3 and 4. The datademonstrate that several isomers of propanediol and butanediol inducemelanogenesis and differentiation of S91 melanoma cells. Both 50 mMpropanediol (PG) or butanediol (BD) resulted in an approximate 1.5-foldincrease of melanogenesis, while 150 mM resulted in about a 2-foldincrease following a single treatment. Whereas 1,2 propanediol (PG-1,2)and (S)-(+)-1,2-Propanediol (PG-S-1,2) resulted in no reduction of cellproliferation at the levels used in this experiment, 150 mM1,3-propanediol (PG-1,3), 2,3-butanediol (BD-2,3) or 1,3-butanediol(BD-1,3) resulted in a reduction of cell numbers by one-third. Inaddition, the butanediols appeared to result in greater differentiationof S91 cells than the propanediols, as evidenced by earlier and greatermorphological changes, and in the case of BD-2,3, a more adherentphenotype. Ethanol (EtOH) had no effect on cells at 340 mM but was toxicat 850 mM as indicated by low cell survival. Ethanol did not inducemelanogenesis at any concentration tested. Glycerol (G) had only aslight effect-on melanogenesis and differentiation at the concentrationstested in this experiment, indicating that triols may be less effectiveinducers of these phenotypes than diols. TABLE 3 Cells (× 10⁶) ugMelanin pg Melanin/Cell Control 0.100 1.17 11.7 1.0% ETOH¹ 0.104 1.1411.0 2.0% ETOH² 0.100 1.25 12.5 5.0% ETOH³ 0.032 0.17 5.3  50 mM PG-1,20.084 1.31 15.6 150 mM PG-1,2 0.072 1.73 24.0  50 mM PG-S-1,2 0.088 1.5117.1 150 mM PG-S-1,2 0.080 2.04 25.5  50 mM PG-1,3 0.064 1.31 20.4 150mM PG-1,3 0.044 1.04 23.6  50 mM G 0.092 1.03 11.2 150 mM G 0.084 1.0913.0  50 mM BD-2,3 0.072 1.12 15.6 150 mM BD-2,3 0.040 0.95 23.8  50 mMBD-1,3 0.064 0.99 15.5 150 mM BD-1,3 0.048 0.87 18.1

[0184] TABLE 4 Morphology Rounded Flattened Trypsinization SpindlyCuboidal Detachment Time Control 100% 3 min 1.0% ETOH 100% 3 min 2.0%ETOH 100% 3 min 5.0% ETOH 100% 3 min  50 mM PG-1,2  75%  25% 3 min 150mM PG-1,2  50%  50% 6 min  50 mM PG-S-1,2  75%  25% 3 min 150 mMPG-S-1,2  50%  50% 6 min  50 mM PG-1,3  75%  25% 3 min 150 mM PG-1,3 50%  50% 6 min  50 mM G 100% 3 min 150 mM G  75%  25% 3 min  50 mMBD-2,3  25%  75% 3 min 150 mM BD-2,3 100% 9 min  50 mM BD-1,3  25%  75%3 min 150 mM BD-1,3 100% 6 min

[0185] Melanogenesis is the most characteristic feature of melanocytedifferentiation (J. Cell Sci. 107:1095-1103, 1994), and, is inverselycorrelated with rate of proliferation in melanoma cell lines (Neoplasia31:545-9, 1984; Biochem. Biophys. Res. Commun. 177:545-50, 1991; Exp.Dermatol. 4:192-198, 1995). As a general rule, increased proliferationcommensurate with dedifferentiation are hallmarks of rapid tumorprogression and a poor prognosis, while decreased proliferation anddifferentiation are indicative of more long-term survival (Introductionto the Cellular and Molecular Biology of Cancer, L. M. Franks and N.Teich, 1987, Oxford University Press). Thus, the ability of the presentcompounds to induce melanogenesis and slow cell growth is indicative oftheir ability to act as chemotherapeutic agents. Induction ofmelanogenesis combined with a reduced rate of cellular proliferation isindicative of induction of differentiation in S91 cells. In addition,the change of cellular morphology from a rounded, spindly appearance toa flattened, cuboidal appearance is further indication ofdifferentiation in S91 cells (Exp. Cell Res. 191:209-218, 1990). Thus,the compounds of the present invention are not only tanning agents, butalso chemotherapeutic agents capable of delaying tumor progression andincreasing long-term survival.

[0186] It should be noted that the effects of propylene glycol(Example 1) and related diols and triols (Examples 1 & 2) on S91 cellsare identical to those resulting from treatment of S91 cells withretinoids; that is, induction of melanogenesis, induction ofdifferentiation, increased adherence, and inhibition of proliferation(Laukharanta, et al., Arch. Dermatol. Res. 277:147-150, 1985). Giventhis similarity of biological responses, it is believed that the agentsdescribed herein are effective in treating those disorders presentlytreated with the retinoids including a variety of forms such aspsoriasis, acne and dermatoses.

EXAMPLE 3

[0187] The same procedures as in Examples 1 and 2 were followed toexamine the effect of additional compounds on melanogenesis in S91cells. The results described in Table 5 show the concentration of anumber of compounds required to induce 2-fold or greater melanization inS91 cells. Many compounds are more potent than those described inExamples 1 and 2. For example, 2,3-pyridinediol was potent at 100 uM;1,4-dioxane-2,3-diol and β-estradiol at 500 um;5-norbornene-2,2-dimethanol at 5 mM; 3,3-dimethyl-1,2-butanediol and1,2-cis-cyclopentanediol at 10 mM; and 2,3-dimethyl-2,3-butanediol at 25mM. All of the compounds listed in Table 5 except 1,4-dioxane-2,3-diol,induced transformation of S91 cells from a rounded bipolar morphology toa flattened cuboidal multipolar morphology concomitant with induction ofmelanogenesis; this indicates their potential usefulness aschemotherapeutic agents that act by inducing differentiation of tumorcells. All of the compounds listed in Table 5 except5-norbornene-2,2-dimethanol, β-estradiol, and 2,3-pyridinediol inducedincreased trypsinization time concomitant with induction ofmelanogenesis; alterations of adherence properties are related tochanges of metastatic potential of tumor cells. TABLE 5 ConcentrationRequired for ≧2-fold Compound Melanin Induction in S91 Cells2,3-Pyridinediol 100 uM 1,4-Dioxane-2,3-Diol 500 uM β-Estradiol 500 uM5-Norbornene-2,2-Dimethanol  5 mM 1,2-cis-Cyclopentanediol  10 mM3,3-Dimethyl-1,2-Butanediol  10 mM 2,3-Dimethyl-2,3-Butanediol  25 mM1,2-trans-Cyclopentanediol  50 mM 2-Methyl-1,3-Propanediol  50 mM2,3-Butanediol 100 mM 1,2-Propanediol 150 mM

[0188] Compounds in addition to those described in Examples 1 and 2,that did not induce significant (≧2-fold increase) melanogenesis in S91cells when tested over a range of concentrations up to a toxic doseincluded: 1-propanol; 2-propanol; oleic acid; 2-phenyl-1,2-propanediol;1,3-cyclohexanediol; tartaric acid; ascorbic acid; Azone®,2-pyrrolidone; D-ribose; 2-deoxy-D-ribose; N-methyl-D-glucamine;hydroxymethyl uracil; and tetrabutylammonium chloride. Of thesecompounds, only 2-pyrrolidone resulted in profound morphologicaldifferentiation of S91 cells, indicating that it may augmentmelanogenesis and/or exert antitumorigenic activity in the absence ofmelanogenesis.

[0189] The PKC inhibitors H7(1-[5-isoquinolinyl-sulfonyl]-2-methyl-piperazine) and D-sphingosinealso induced melanogenesis in S91 cells. In addition, these PKCinhibitors enhanced melanogenesis induced by propylene glycol in S91cells. These results indicate that propylene glycol does not inducemelanogenesis by induction of PKC, or require PKC for induction ofmelanogenesis.

EXAMPLE 4

[0190] Normal human epidermal melanocytes (NHEMs) were examined forinduction of melanogenesis using cells and media from CloneticsCorporation (San Diego, Calif.). Cells were cultured exactly asspecified by the supplier. Based on induction of a 1.5-fold increase ofmelanin in NHEMs, the most potent compound examined was2,3-pyridine-diol at 200 uM, followed by 5-norbornene-2,2-dimethanol at≦5 mM, 3,3-dimethyl-1,2-butanediol at 12.5 mM, and2,3-dimethyl-2,3-butanediol and 1,2-cis-cyclopentanediol at 50 mM (Table6). D-Ribose was inactive in NHEMs when tested over a range ofconcentrations up to a toxic dose. These results show that compounds ofthe present invention that exhibit activity in S91 cells, also exhibitactivity in normal human melanocytes. TABLE 6 Concentration Required for≧1.5-fold Compound Melanin Induction in NHEMs 2,3-Pyridinediol  200 uM5-Norbornene-2,2-Dimethanol   5 mM 3,3-Dimethyl-1,2-Butanediol 12.5 mM1,2-cis-Cyclopentanediol   50 mM 2,3-Dimethyl-2,3-Butanediol   50 mM1,2-Propanediol  150 mM

EXAMPLE 5

[0191] Compounds were tested for melanogenic activity in vivo byapplication to American short-haired guinea pigs. Treatment sites werecreated by removal of fur using Nair® brand depilatory. Compounds wereapplied in 25 μl volumes twice per day for 5 days to each treatment spotas indicated in Table 7. In the Table, the numbers presented are therelative melanogenesis rating (mean±SE), and are arranged according tothe relative location on the animal, with the head being to the left andthe tail being to the right. Propylene glycol (PG=13.6M), 2,3-butanediol(2,3-BD=10.95M), and 1,2-cis-cyclopentanediol (1,2-cs-CPD=10.7M) wereapplied as full strength solutions. 3,3-dimethyl-1,2-butanediol(3,3-M-1,2-BD) was applied as a 4M solution dissolved in ethanol. Twoweeks following cessation of treatments, the degree of pigmentation wassubjectively rated according to the following scale: 0 no change 0.5slight darkening, not easily discernible 1 slight darkening, easilydiscernible 2 moderate, even darkening 3 substantial, even darkening 4profound, even darkening

[0192] The results presented below showed that there was a progressivediminution of response to tanning agents from head to tails of animals.The magnitude of this diminished response was 3- to 4-fold. Thus,comparisons between treatment compounds were done relative to similarlocations on the body of guinea pigs. Propylene glycol resulted insignificant melanogenesis relative to depilitory treated controlslocated at the same relative body position. 2-methyl-1,3-propyleneglycol and 2,3-butanediol were only slightly better melanogenic agentsthan propylene glycol. However, 3,3-dimethyl-1,2-butanediol and1,2-cis-cyclo-pentanediol resulted in 4.5-fold and 5.5-fold greatermelanogenesis than PG applied at similar body locations. TABLE 7Treatment Head <--------------------------------> Tail a b c d PG, 5Days (n = 6): 1.04 ± 0.21 0.83 ± 0.17 0.25 ± 0.09¹ 0.33 ± 0.16¹ 5 days(n = 3): 2-M-PG 2,3-BD 2-M-PG 2,3-BD 1.25 ± 0.52 1.33 ± 0.17² 0.58 ±0.08² 0.25 ± 0.14 5 Days (n = 3): Nair PG 3,3-M-1,2-BD 1,2-cs-CPD 0²0.50 ± 0.25 1.16 ± 0.66² 1.83 ± 0.33²

[0193] In order to minimize the effects of dimunition of response fromhead to tails of animals, all future experiments were done using onlytreatment spots located towards the tails of animals (c and d in Table7). Deemed as additionally beneficial, in this area of the animaldifferences of responsiveness to strong and weak inducers ofpigmentation, as deduced from cell culture, were greatest. Comparison ofthe pigmentation ratings of these treatment spots showed the followingdescending order of induction: 8.7M 1,2-cis-cyclopentanediol(1,2-cs-CPD)>4M 3,3-di-methyl-1,2-butanediol (3,3-M-1,2-BD)>a mixture of8.5M 1,2-propylene glycol (1,2-PG)/1M 5-norbornene-2,2-dimethanol(5-NBene-2,2-DM)/2% 2-pyrrolidone (2-P; a penetration enhancer)>1M5-NBene-2,2-DM/2% 2P,>11.3M 2-methyl-1,3-propylene glycol (2-M-1,2-PG)(Table 8; FIG. 1A: untreated; 1B: 10.6M 1,2-PG/2% 2-P; 1C: 8.7M1,2-cs-CPD; 1D: 1M 5NBene-2,2-DM/8.5M 1,2-PG/2% 2-P). In this region ofthe animals, responses to 13.61M 1,2-PG; 10.6M 1,2-PG/2% 2P, and 11M2,3-dimethyl-2,3-butanediol were not significantly different fromcontrol (Nair or 2% 2P treated) spots. Pigmentation ratings werecorrected for background (control treatment spots), normalized to 1M toaccount for the different amounts of each agent applied, and thennormalized to results for 1,2-PG (Table 8). This comparison showed thatthe descending order of induction was5-NBene-2,2-DM>1,2-cs-CPD>2-M-1,3-PG, and, that using 1,2-PG as carrierfor 5-NBene-2,2-DM (FIG. 1D) increased responsiveness to this compound.It is anticipated that further improvements in formulation willadditionally improve responsiveness to 5-NBene-2,2-DM and othercompounds in this invention. Biopsies results (FIG. 1) showed thatinduction of melanogenesis was marked by deposition of melanin inkeratinocytes, in some cases with formation of “supranuclear caps”(arrows, FIGS. 1C & 1D) indicative of induction of true naturalUV-protective melanogenesis (Gates, R. R., and A. A. Zimmerman, 1953 J.Invest. Dermatol. 21:339-348), and a complete absence of inflammation,fibrosis or any other form of tissue damage. TABLE 8 PigmentationBackground Normalized Normalized Treatment Rating Corrected to 1M to1,2-PG No Penetration Enhancer Nair 0.08 ± 0.05 0 (n = 6) 13.61M 0.29 ±0.09 0.21 ± 0.03 0.015 ± 0.002 1.0 ± 0.1 1,2-PG  (n = 12) 11.0M 0.25 ±0.14 0.17 ± 0.09 0.015 ± 0.008 1.0 ± 0.6 2,3-M-2, (n = 3) 3-BD 11.3M 0.58 ± 0.08* 0.50 ± 0.07 0.044 ± 0.006 2.9 ± 0.4 2-M-1, (n = 3) 3-PG8.7M  1.89 ± 0.27* 1.75 ± 0.25 0.202 ± 0.029 13.5 ± 1.9  1,2-cs- (n = 9)CPD 4.0M  1.17 ± 0.44* 1.09 ± 0.41 0.272 ± 0.102 18.1 ± 6.8  3,3-M-1, (n= 3) 2-BD

[0194] Penetration Enhancer 2% 2-Pyrrolidone 2P 0.17 ± 0.08 0 (n = 6)10.6M 0.33 ± 0.05 0.16 ± 0.02 0.015 ± 0.002  1.0 ± 0.15 1,2-PG/2P (n =6) 1.0M  0.66 ± 0.05* 0.49 ± 0.04 0.490 ± 0.037 32.7 ± 2.55-NBene-2,2-DM/2P (n = 6) 8.5M  1.00 ± 0.13* 0.83 ± 0.11 0.670 ± 0.087¹44.7 ± 5.8 1,2-PG/2P/1.0M 5-NBene-2,2-DM (n = 6)

EXAMPLE 6

[0195] Compounds were examined for their ability to induce tyrosinaseactivity in S91 mouse melanoma cells. Tyrosinase is the rate limitingenzyme in the melanogenic pathway. Its measurement provides a highlyspecific and sensitive indication of degree of induction ofmelanogenesis by test compounds. All cell culture conditions andtreatments were as described above in Examples 1-3. Followingtreatments, cells were trypsinized, counted by Coulter, pelleted bycentrifugation at 1000×g, and analyzed for tyrosinase activity usingmodifications of previously described procedures (Pomerantz, S. H.,1966, J. Biol. Chem. 241:161-168; Jara, et al., 1988, Pigment Cell Res.1: 332-339.). Briefly, cell pellets were solubilized by sonicating for 5seconds in 600 ul 50 mM phosphate buffer pH 6.8 containing 0.5%Triton-X100, followed by vortexing, incubation on ice for 30 min, andthen revortexing. From this, 200 ul aliquots were combined with 200 ulof reaction mixture containing either 75 uM tyrosine, 75 uM L-Dopa, and2 uCi L-[3,5⁻³H]Tyrosine in 50 mM NaPO₄ pH 6.8 (L-Dopa+), or, 75 uMtyrosine, and 2 uCi L-[3,5⁻³H]Tyrosine in 50 mM NaPO₄ pH 6.8 (L-Dopa−)and incubated 1 hr at 37° C. Reactions were stopped by addition of 400ul 10% activated charcoal in 0.1N HCl and incubation on ice for 15 min.This mixture was centrifuged at 17,300×g for 5 min, and 400 ulsupernatant was then filtered through a 0.22 uM GV Durapore centifugalfilter unit (Millipore) by centrifuging at 17,300×g for 5 min. Filtratewas added to 4 ml Fisher Plus scintillation fluid and counted on aHewlett Packard scintillation counter. Tyrosinase activity wascalculated as dpm/hr/ug protein and dpm/hr/10³ cells. Each sample wasanalyzed with and without L-Dopa, a necessary cofactor for tyrosinase(Pomerantz, S. H., 1966, J. Biol. Chem. 241:161-168; McLane, et al.,1987, Biochem. Biophys. Res. Commun. 145:719-725). All reportedtyrosinase values are exclusive of counts that occurred in buffer blanksand L-dopa negative aliquots. Protein was determined on aliquots of celllysate, extracellular particulate lysate or media by the BradfordCoomassie Blue method (Bradford, 1967, Anal. Biochem. 72:248-254) usingBio-Rad Protein Assay Kit I.

[0196] Results (Table 9; mean±SE) show that 3,3-dimethyl-1,2-butanediol(3,3-M-1,2-BD) and 5-norbornene-2,2-dimethanol (5-NBene-2,2-DM) resultin the greatest induction of tyrosinase on both a cellular and proteinbasis. Although 100 uM 2,3-pyridinediol (2,3-Pyd) induced 2-foldincreases of melanin (Example 3, Table 5), even 500 uM 2,3-Pyd inducedonly low levels of tyrosinase relative to that induced by 5 mM5-NBene-2,2-DM or 3,3-M-1,2-BD, and, higher levels of 2,3-Pyd were toxic5-NBene-2,2-DM and 3,3-M-1,2-BD are nontoxic at concentrations thatinduce much higher levels of tyrosinase, and thus are preferred agentsfor induction of melanogenesis in this embodiment. Since 5-NBene-2,2-DMinduces nearly equivalent levels of tyrosinase at 5-fold lowerconcentrations than 3,3-M-1,2-BD, it is particularly preferred. IBMX(3-isobutyl-1-methylxanthine) is well known to those in the art aspotent inducer of melanogenesis and tyrosinase, and is provided as apositive control. TABLE 9 dpm/hr dpm/hr ug Sample #/Treatment 10³ CellsProtein Control (n = 4)  40 ± 6  184 ± 27 300 mM PG-1,2 (n = 4)  292 ±104 1003 ± 370  25 mM 3,3-M-1,2-BD (n = 2) 1211 ± 38 1746 ± 220  50 mM1,2-cs-CPD (n = 2)  276 ± 16  925 ± 53  5 mM 5-NBene-2,2-DM (n = 4)  707± 54 1643 ± 105  0.5 mM 2,3-Pyd (n = 2)  142 ± 8  160 ± 19  0.1 mM IBMX(n = 2)  765 ± 53 2161 ± 41

[0197] Structure activity studies with 5-NBene-2,2-DM and relatedcompounds indicate that norbornane-2,2-dimethanol (NBane-2,2-DM) hasequivalent potency for induction of tyrosinase in S91 cells (FIG. 2).Thus, NBane-2,2-DM is equivalently preferred with 5-NBene-2,2-DM. Lesserinduction of tyrosinase in S91 cells was induced in descending order by2-Norbornanemethanol (2-NBaneM), 2,3-cis/exo-Norbornanediol(2,3-c/e-NBaneD), α-Norborneol (α-NBane-ol), and Norbornane (NBane).Since even NBane results in 2-fold induction of tyrosinase relative tountreated or ethanol (ETOH) treated control S91 cells, it is included asa component of this invention. In addition, since NBane inducesmelanogenesis, it is contemplated that all compounds containing NBane asa component of their structure may induce melanogenesis. In addition,compounds containing Norbornene (NBene) or any other unsaturatedcompound derived form norbornane are expected to induce melanogenesis.Thus, any saturated or unsaturated compound derived from or related tonorbornane is included as a component of this invention, including butnot limited to compounds derived from bornane, pinane, camphene andcamphor.

[0198] Neither the highly specific protein kinase A (PKA) inhibitor H-89(N-[2-(p-bromocinnamylamino)-ethyl]-5-isoquinolinesulfinamide.2HCl;Chijiwa, et al., 1990, J. Biol. Chem. 265:5267-5272), nor the highlyspecific protein kinase C (PKC) inhibitor GF109203X(Bisindolylmaleimide; Toullec, et al., 1991, J. Biol. Chem.266:15771-15781) inhibited induction of tyrosinase by 5-NBene-2,2-DM(Table 10). Thus, similar to results described for 1,2-propanediol inExample 3,5-NBene-2,2-DM and related compounds are unlikely to act viaactivation of PKC pathways, which have been described as important forinduction of melanogenesis by diacylgerols (Allan, et al., 1995, J.Invest. Dermatol. 105:687-692; Gilchrest, et al., 1996, Photochem.Photobiol. 63:1-10). Nor are 5-NBene-2,2-DM or related compounds likelyto act via activation of PKA pathways, described as important forinduction of melanogenesis by IBMX (Fuller, et al., 1993, Ann. NY Acad.Sci. 690:302-319; Fuller, et al., 1996, Pigment Cell Res. S5:65).Furthermore, addition of catalase to the cell culture media did notinhibit the action of 5-NBene-2,2-DM, indicating that unlike L-Dopa andDopac, this and related compounds are unlikely to induce melanogenesisvia generation of hydrogen peroxide or other reactive oxygen species(Karg, et al., 1989, Acta Derm. Venereol. 69:521-524; Karg, et al.,1991. J. Invest. Dermatol. 96:224-227; Karg, et al., 1993, J. Invest.Dermatol. 100:209S-213S). TABLE 10 Tyrosinase dpm/hr/ug Relative toProtein Control Control 398 1   5 mM 5-NBene-2,2-DM 3273 8.2X   1 uMH-89 507 1.3X  10 uM H-89 1236 3.1X   1 uM H-89/ 4624 11.6X   5 mM5-NBene-2,2-DM  10 uM H-89/ 3093 7.8X   5 mM 5-NBene-2,2-DM 0.1 uMGF109203X 1025 2.6   1 uM GF109203X 2407 6.1X 0.1 uM GF109203X/ 467911.8X   5 mM 5-NBene-2,2-DM   1 uM GF109203X/ 6531 16.4X   5 mM5-NBene-2,2-DM  500 Units Catalase/ml 745 1.9X 1000 Units Catalase/ml691 1.7X  500 Units Catalase/ml/ 2796 7.0X   5 mM 5-NBene-2,2-DM 1000Units Catalase/ml/ 4778 12.0X   5 mM/5-NBene-2,2-DM

EXAMPLE 7

[0199] Tyrosinase was measured in normal human epidermal melanocytes(NHEM) using procedures identical to those described for S91 cells(Example 6), except that media from day treatment periods was retainedand centrifuged at 200×g, 1600×g, or 17,300×g for analysis of tyrosinaseactivity in the extracellular exported melanosomal particulate fraction,and in the resultant supernatant media fraction. In some cases (Table11), tyrosinase was also measured by an in situ assay whereinradiolabelled tyrosine was added directly to freshly replaced media ofNHEM for a period of 24 hrs following a 5 day treatment period(Abdel-Malek, et al., 1992, J. Cell. Physiol. 150:416-425). Resultsshowed that 5 mM 5-NBene-2,2-DM induced tyrosinase to a greater extentin the in situ assay, in cells, in extracellular particulate melanosomalfractions, and in the media of NHEM than did 25 mM 3,3-M-1,2-BD (Table11). Both 5 mM 5-NBene-2,2-DM and 25 mM 3,3-M-1,2-BD induced moretyrosinase in each of these assays and fractions than did 1,2-PG. IBMX(3-isobutyl-1-methyl-xanthine) provided as a positive control, inducedas much tyrosinase as 5 mM 5-NBene-2,2-DM in the in situ assay, but lessin cellular, extracellular particulate and media fractions (Table 11).TABLE 11 Tyrosinase dpm/hr/10³ Cells In 200 g 17300 g* Situ CellularPartic Partic Media** Control 16.8 10259  244  97 1457 85 mM ETOH 15.010201  442 132 1654 (1.00X) (1.00X) (1.00X) (1.00X) (1.00X) 300 mM1,2-PG 16.8 10247  433 102 1864 300 mM 1,2-PG 17.2 10875  923 241 2123(1.07X) (1.03) (1.98X) (1.50X) (1.28X) 25 mM 20.5 11728 1646 536 54953,3-M-1,2-BD 25 mM 21.0 11730 2226 425 3056 3,3-M-1,2-BD (1.31X) (1.15X)(5.64X) (4.20X) (2.75X) 5 mM 24.5 13838 6447 493 4164 5-NBene-2,2-DM 5mM 25.4 14716 6291 473 4639 5-NBene-2,2-DM (1.57X) (1.40X) (18.6X)(4.22X) (2.83X) 0.1 mM IBMX 25.3 10910 2189 220 2698 0.1 mM IBMX 26.111737 1834 260 2935 (1.62X) (1.11X) (5.86X) (2.10X) (1.81X)

[0200] Further studies using NHEM demonstrated that, similar to resultsfor S91 cells (FIG. 2), compounds related to 5-NBene-2,2-DM may beinducers of tyrosinase (Table 12). For example, 2-norbornanemethanol(2-NBaneM) resulted in induction of tyrosinase at levels equivalent to5-NBene-2,2-DM in NHEM both from a white adult donor and a blackneonatal donor (Table 12). Thus, similar to S91 cells (Example 6), allnorbornane-related compounds are contemplated to induce tyrosinase inNHEM and are thereby embodied in this invention. TABLE 12 Tyrosinasedpm/hr/10³ cells White-Adult-NHEM In Situ Cellular Media¹ Control 5.56(1.00X) 13992 (1.00X) 36.3 (1.00X) 1 mM 5-NBene-2,2-DM 6.27 (1.13X)12740 (0.91X) 29.9 (0.82X) 5 mM 5-NBene-2,2-DM 5.81 (1.04X) 18467(1.32X) 53.1 (1.46X) 1 mM 2-NBaneM 7.05 (1.27X) 15257 (1.09X) 29.2(1.11X) 5 mM 2-NBaneM 6.18 (1.11X) 16077 (1.15X) 48.1 (1.33X) dpm/hr/10³cells Black-Neonatal-NHEM In Situ Cellular Media Control 12.5 (1.00X) 9856 (1.00X) 11.1 (1.00X) 1 mM 5-NBene-2,2-DM 13.9 (1.11X) 10679(1.08X) 26.8 (2.41X) 5 mM 5-NBene-2,2-DM 14.1 (1.13X) 15398 (1.56X) 33.2(2.99X) 1 mM 2-NBaneM 12.1 (0.97X) 10863 (1.10X) 18.7 (1.68X) 5 mM2-NBaneM 12.8 (1.02X) 17397 (1.77X) 37.3 (3.36X)

EXAMPLE 8

[0201] Similar to results for S91 cells treated with diols (Examples 1and 2), treatment of normal human epidermal melanocytes (NHEM) with 5 mM5-NBene-2,2-DM resulted in morphological changes indicative ofdifferentiation. In the case of NHEM, induction of differentiation wasmarked by conversion of cells from a bipolar phenotype to amultidendritic phenotype (compare untreated NHEM in FIG. 3A with 5 mM5-NBene-2,2-DM treated NHEM in FIG. 3B). Additionally, the length ofdendrites was increased approximately 2-3-fold following treatment with5 mM 5-NBene-2,2-DM, and there was an increase in the number ofsecretatory vesicles at the termini of dendrites (arrows in FIGS. 3A and3B). Electron microscopic analysis indicated that the extracellularparticulate fraction secreted into the media from NHEM was comprisedalmost exclusively of stage III and IV melanosomes (arrows showlongitudinal view and arrowheads show cross-sectional view in FIGS. 3Cand 3D). Increased secretion of melanosomes resulting from treatmentwith 5 mM 5-NBene-2,2-DM was reflected in increased extracellularparticulate tyrosinase activity (Example 7, Table 11).

[0202] It is well known that ultraviolet irradiation of skin results inincreased dendricity of melanocytes and increased transport ofmelanosomes from the ends of dendritic processes to neighboringkeratinocytes (Jimbow, et al., Biology of Melanocytes, pp. 261-289, In:Dermatology in General Medicine, eds: Fitzpatrick, et al., McGraw-Hill,1994). Thus, secretion of melanosomes from melanocytes treated with5-NBene-2,2-DM appears to parallel the physiological processes inducedby sunlight in skin.

EXAMPLE 9

[0203] Highly specific inhibitors of the cAMP/PKA (protein kinase A) orPKC (protein kinase C) pathways do not inhibit induction ofmelanogenesis by 5-NBene-2,2-DM in S91 cells (Example 6, Table 10).However, each of the nitric oxide (NO) scavenger PTIO(2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide), the cyclicguanosine monophosphate (cGMP) inhibitor LY83583(6-anilino-5,8-quinolinequinone), and the PKG (protein kinase G)inhibitor KT58223 reduce induction of melanogenesis by 5-NBene-2,2-DM inS91 cells (Table 13). These results demonstrate that induction ofmelanogenesis by 5-NBene-2,2-DM occurs by the NO/cGMP/PKG pathway.Furthermore, results are similar to those obtained for ultravioletradiation wherein induction of melanogenesis did not occur via eitherthe cAMP/PKA or PKC pathways (Friedmann and Gilchrest, 1987, J. Cell.Physiol 133:88-94; Carsberg, et al., J. Cell. Sci. 107:2591-2597), butrather occurred via the NO/cGMP/PKG pathway (Romero-Graillet, et al.,1996, J. Biol. Chem. 271:28052-28056; Romero-Graillet, et al., 1997, J.Clin. Invest. 99:635-642). Moreover, unlike IBMX(3-isobutyl-1-methylxanthine) and MSH (melanocyte stimulating hormone)which induce melanogenesis by the cAMP/PKA pathway (Wintzen andGilchrest, 1996, J. Invest. Dermatol. 106:3-10; Fuller, et al., 1993,Ann. NY Acad. Sci. 690:302-319), and DAG (diacylglycerol) which inducesmelanogenesis by the PKC pathway (Allan, et al., 1995, J. Invest.Dermatol. 105:687-692), 5-NBene-2,2-DM induces melanogenesis by theNO/cGMP/PKG pathway similar to ultraviolet radiation.

[0204] It has been previously demonstrated that a variety of aliphaticand alicyclic diols including 5-norbornene-2,2-dimethanol(5-NBene-2,2-DM) induce melanogenesis in S91 cells (Examples 1-3). Theresults presented in Table 15 show that induction of tyrosinase (therate-limiting enzyme in melanogenesis) by 5-NBene-2,2-DM is not blockedby highly specific inhibitors of the PKC and PKA pathways. In fact,treatment of S91 cells with either the highly specific PKA inhibitorH-89 (Chijiwa, et al., 1990, J. Biol. Chem. 265:5267-5272), or thehighly specific PKC inhibitor GF109203X (Toullec, et al., 1991, J. Biol.Chem. 266:15771-15781) resulted in augmentation of basal and5-NBene-2,2-DM-induced tyrosinase levels (Table 15). Thus,5-NBene-2,2-DM does not appear to act via either the PKC or PKApathways.

[0205] In contrast, both the nitric oxide (NO) scavenger PTIO(2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide), the cyclicguanosine monophosphate (cGMP) inhibitor LY83583(6-anilino-5,8-quinolinequinone), and the PKG (cGMP-activated proteinkinase) inhibitor KT5823 reduced induction of melanogenesis by5-NBene-2,2-DM in S91 cells (Table 16). These results demonstrate thatinduction of melanogenesis by 5-NBene-2,2-DM occurs by the NO/cGMP/PKGpathway.

[0206] Previously, it has been demonstrated that NO donors can stimulatemelanogenesis in normal human melanocytes (Romero-Graillet, et al.,1996, J. Biol. Chem. 271). Results presented here demonstrate that5-NBene-2,2-DM can stimulate melanogenesis with an efficacy equivalentor greater than that of NO donors, even though 5-NBene-2,2-DM has noability to donate NO. Since induction of melanogenesis by 5-NBene-2,2-DMoccurs by the NO/cGMP/PKG pathway, 5-NBene-2,2-DM must directlystimulate NO synthesis within cells.

[0207] These results demonstrate that stimulation of NO synthesis andthe cGMP/PKG pathway by 5-NBene-2,2-DM provides an efficient alternativeto stimulation of this pathway by NO donors. Thus, 5-NBene-2,2-DM andrelated compounds described in this invention will serve as alternativetherapeutics for treatment of a variety of diseases mediated byperturbations of the NO/cGMP/PKG pathway. TABLE 13 % of dpm/hr/ 5-NBene-Induction 10³ cells 2,2-DM NO/PKG Inhibitors - Experiment 1 5 mM5-NBene-2,2-DM (n = 4) 5018 ± 415¹ 100% 5 mM 5-NBene-2,2-DM/ 3703 ± 262 74% 20 uM PTIO² (n = 2) 5 mM 5-NBene-2,2-DM/ 1528 ± 190  31% 0.5 uMKT5823³ (n = 2) ¹ X ± SE ² PTIO: Nitric oxide scavenger ³ KT5823: PKGinhibitor NO/PKG Inhibitors - Experiment 2 5 mM 5-NBene-2,2-DM (n = 4)5640 ± 323 100% 5 mM 5-NBene-2,2-DM/ 4078 ± 429  72% 20 uM PTIO² (n = 2)5 mM 5-NBene-2,2-DM/ 3351 ± 994  59% 40 uM PTIO (n = 2) 5 mM5-NBene-2,2-DM/ 2940 ± 261  52% 0.5 uM KT5823³ (n = 2) 5 mM5-NBene-2,2-DM/ 1.0 uM KT5823 (n = 2) 1688 ± 324  30% ² PTIO: Nitricoxide scavenger ³ KT5823: PKG inhibitor cGMP Inhibitor - Experiment 3 5mM 5-NBene-2,2-DM (n = 4) 6388 ± 460¹ 100% 5 mM 5-NBene-2,2-DM/ 1389 ±64  22% 0.1 uM LY83583⁴ (n = 2) 5 mM 5-NBene-2,2-DM/  300 ± 84  5% 0.2uM LY83583 (n = 2)

EXAMPLE 10

[0208] Studies with the L-arginine analog S-ethylisothiourea (Garvey, etal., 1994, J. Biol. Chem. 269:26669-26676; Southern, et al., 1995, Br.J. Pharmacol. 114:510-516), a competitive inhibitor of nitric oxidesynthase at the L-arginine binding site, also the contention that5-norbornene-2,2-dimethanol acts via the nitric oxide pathway. Treatmentwith S-ethylisothiourea (S-EITU) resulted in a dose-response diminutionof tyrosinase activity in S91 cells, with complete ablation oftyrosinase activity at 1000 nM S-EITU (Table 14). TABLE 14 Tyrosinasedpm/hr/10³ Cells Control  47 86 mM ETOH  46 5 mM 5-NBene-2,2-DM 4515 5mM 5-NBene-2,2-DM 4247 Avg. 4381 5 mM 5-NBene-2,2-DM/50 nM S-EITU 5186 5mM 5-NBene-2,2-DM/100 nM S-EITU 4646 5 mM 5-NBene-2,2-DM/250 nM S-EITU3758 5 mM 5-NBene-2,2-DM/500 nM S-EITU 1055 5 mM 5-NBene-2,2-DM/750 nMS-EITU  357 5 mM 5-NBene-2,2-DM/1000 nM S-EITU Not Done

EXAMPLE 11

[0209] As a continuance of the structure activity studies described inExample 6 (FIG. 2), a variety of norbornane derivatives and relatedmonocyclic or aliphatic derivatives were examined for melanogenicactivity (Tables 15 and 16). Although many of these agents possessedsignificant melanogenic activity, only 2-hydroxy-2-norbornanemethanoland 1-(exo & endo-2-norbornyl-)-propan-1,2-diol induced levels oftyrosinase that approached the maximal levels induced by5-norbornene-2,2-dimethanol (Table 16).

[0210] The results presented in Table 16 demonstrate that severaldifferent types of norbornane derivatives including triols, acetates,acetate esters, carboxylic acids, and formates possess melanogenicactivity. As shown previously in Example 6, some of this activity isembodied within the norbornane structure itself, since 1 or 5 mMnorbornane resulted in 2-fold induction of tyrosinase (FIG. 2). Theseresults further substantiate the claims herein, that any compoundderived from norbornane is expected to be a melanogenic agent, and istherefore included in this invention.

[0211] Low levels of melanogenic activity were also exhibited bymonocyclic dimethanol compounds and a noncyclic dimethanol-containingcompound (Table 16). These results demonstrate that dimethanol groupsembody low levels of melanogenic activity, even in the absence of thebicyclic ring structure of norbornane. These results, combined with thefinding that 2-norbornanemethanol exhibits significant melanogenicactivity (FIG. 2; Table 12), demonstrate that any compound containingone or more methanol groups has the potential to be a melanogenic agent,and these are therefore also included in this invention. TABLE 15 FoldInduction of Tyrosinase Relative to Controls 1 mM 2 mM 5 mM5-norbornene-2,2-dimethanol 4.0X 12.9X 41.9X 2,5 & 6,7-norbornanetriol¹5.1X  5.1X  ND² mono- & di-acetate 1.5X  3.0X ND 2,5 &6,7-norbornanetriol 2-norbornaneacetic acid 4.6X ND 12.9X5-norbornene-2,3-cis/endodicarboxylic 2.2X ND  1.0X acid ±exo-2-norbornyl formate 2.7X ND  2.3X

[0212] TABLE 16 Fold Induction of Tyrosinase Relative to Controls 0.5 mM1 mM 2.5 mM 5 mM 5-norbornene-2,2-dimethanol 2.8X 3.6X 14.5X 61.6X2,7-norbornanediol ND¹ 0.8X 1.6X  3.5X 2-hydroxy-2-norbornanemethanol ND9.2X 15.5X 45.0X 1-(exo & endo-2-norbornyl-)- 2.2X 4.2X 48.5X  2.0Xpropan-1,2-diol methyl-5-norbornene-2,3- 6.0X 8.1X 0.8X  NA² dimethanol1,2-cis-cyclohexanedimethanol ND 0.9X 6.1X NA3-cyclohexane-1,1-dimethanol ND 1.4X 2.3X  7.0X1,4-cyclohexanedimethanol ND 1.2X 1.7X  3.2X pentaerylthritol ND 1.2X1.9X  1.5X

EXAMPLE 12

[0213] Further studies using S91 cells and the methods described inExample 6 showed that2,3-cis/exo-pinanediol([1R,2R,3S,5R]-[−]-pinanediol) had greatermelanogenlc activity than 5-norbornene-2,2-dimethanol when tested over arange of concentrations (FIG. 5). 2,3-cis/exo-pinanediol induced2.6-fold more tyrosinase activity than 5-norbornene-2,2-dimethanol whentested at 500 uM, 5.2-fold more at 1 mM, and 7.3-fold more at 2.5 mM(calculated from data in FIG. 5).

[0214] In a related experiment, nitric oxide was measured in cell-freemedia from S91 cells following treatment with a range of concentrationsof 2,3-cis/exo-pinanediol or 5-norbornene-2,2-dimethanol for 4 days. Inbiological fluids, nitric oxide is converted into nitrite and nitratewith seconds of production. Therefore, nitric oxide is measured by firstconverting nitrate to nitrite using nitrate reductase, followed byaddition of Greiss reagent to detect nitrite as optical density at 550nm (Moshage, et al., 1995, Clin. Chem. 41:892-896; Schmidt, et al.,1995, Biochemica 2:22)). Results of this experiment showed that2,3-cis/exo-pinanediol is a more potent inducer of nitric oxidesynthesis than 5-norbornene-2,2-dimethanol (Table 17). Moreover, therelative melanogenic potency of 2,3-cis/exo-pinanediol and5-norbornene-2,2-dimethanol shown in FIG. 5 paralleled the relativepotency of these compounds with regards to induction of nitric oxide(Table 17). These results in combination with those given in Example 9indicate that similar to induction of melanogenesis by ultravioletirradiation (Romero-Graillet, et al., 1996, J. Biol. Chem.271:28052-28056; Romero-Graillet, et al., 1997, J. Clin. Invest.99:635-642), induction of melanogenesis by diols occurs via the nitricoxide pathway. It follows that measurement of induction of nitric oxide,cGMP or PKG may provide biochemically relevant screening assays forcompounds that may be melanogenic. Thus, the utilization of these assaysto screen compounds for melanogenic activity is claimed in the presentinvention.

[0215] In addition to being a more potent inducer of melanogenesis andnitric oxide (FIG. 4 and Table 17), 2,3-cis/exo-pinanediol was also amore potent inducer of cell cycle arrest than5-norbornene-2,2-dimethanol (Table 17). As discussed in Example 2,induction of melanogenesis in association with cell cycle arrest isindicative of induction differentiation of melanoma cells. Thisindicates that 2,3-cis/exo-pinanediol may have even greater utility than5-norbornene-2,2-dimethanol for use as a chemotherapeuticdifferentiation agent for treatment of melanoma and other types ofcancers. TABLE 17 nmoles NO/ Cells (× 10⁶) uM NO 10⁶ Cells Untreated0.409 ± 0.037 1.74 ± 0.39 4.36 ± 1.16   1 mM 5-NBene-2,2-DM¹ 0.423 ±0.052 4.80 ± 0.32 11.6 ± 1.1* 2.5 mM 5-NBene-2,2- 0.269 ± 0.040* 5.46 ±0.32 21.4 ± 3.7* DM   5 mM 5-NBene-2,2-DM 0.090 ± 0.011* 6.36 ± 0.1272.9 ± 10.5* 0.5 mM 2,3-cs/ex-PD² 0.325 ± 0.002 3.54 ± 0.06 10.9 ± 0.2*  1 mM 2,3-cs/ex-PD 0.258 ± 0.010* 6.36 ± 1.56 24.5 ± 5.4* 2.5 mM2,3-cs/ex-PD 0.099 ± 0.014* 12.6 ± 0.5  131 ± 15*   5 mM 2,3-cs/ex-PD0.064 ± 0.006* 11.0 ± 1.1  174 ± 15*  85 mM ETOH³ 0.454 ± 0.036 3.18 ±0.49 7.04 ± 1.00

EXAMPLE 13

[0216] In studies using the guinea pig model identical to that describedin Example 5,2,3-cis/exo-pinanediol([1R,2R,3S,5R]-[−]-pinanediol)exhibited 2- to 4-fold more melanogenic activity than equivalentconcentrations of 5-norbornene-2,2-dimethanol when compared usingtreatment spots in the posterior half of animals (c and d in Table 18and FIG. 5). In FIG. 5, a, b, c and d indicate treatment spots thattransverse the anterior-posterior axis along the backs of guinea pigs.FIG. 5A, top row, shows spots a-d treated with 50% ETOH; FIG. 5A, bottomrow, shows spots a-d treated with 8.7M 1,2-cis-cyclopentanediol in 20%ETOH; FIG. 5B, top row, shows spots a-d treated with 1M5-norbornene-2,2-dimethanol in 8.5M propylene glycol, 20% ETOH, and 2%2-pyrrolidone; and FIG. 5B, bottom row, shows spots a-d treated with 1M2,3-cis/exo-pinanediol.

[0217] Whereas 5-norbornene-2,2-dimethanol required formulation in 8.5Mpropylene glycol with 2% 2-pyrrolidone to enable penetration of skin andinduction of melanogenesis (see Example 5 and Tables 8 and 18),2,3-cis/exo-pinanediol induced pigmentation when formulated in only 50%ethanol (Table 18). Biopsies show that similar to induction ofmelanogenesis by 1,2-cis-cyclopentanediol (FIGS. 1C and 6B) and5-norbornene-2,2-dimethanol (FIGS. 1D and 6C), induction ofmelanogenesis by 2,3-cis/exo-pinanediol FIG. 6D) was characterized byproliferation of melanocytes in the basal layer of the epidermis anddistribution of melanin throughout the epidermis. Biopsies from skintreated with 50% ethanol (ETOH) exhibited no such response (FIG. 6A).TABLE 18 Pigmentation Ratings¹ of Treated Spots Located Anterior (a) toPosterior (d) on Guinea Pigs a b c d 50% ETOH 0.67 ± 0.12 0.25 ± 0.160.08 ± 0.08 0.04 ± 0.04 8.7 M 1,2-cis- 1.83 ± 0.17* 1.92 ± 0.43* 1.92 ±0.20* 1.58 ± 0.24* CPD²   1 M 5- 1.75 ± 0.43* 1.25 ± 0.21* 0.83 ± 0.15*0.29 ± 0.08* NBene-2,2- DM³   1 M 2,3- 2.00 ± 0.20* 1.75 ± 0.17* 1.75 ±0.17* 1.04 ± 0.32* cs/ex PD⁴

EXAMPLE 14

[0218] As a continuance of structure activity studies, a variety ofpinanediol derivatives and related monocyclic derivatives were examinedfor melanogenic activity using the S91 cell line and procedures foranalysis of tyrosinase described in Example 6. All of the compoundsexamined herein were either bicyclic- or monocyclic-monoterpenes (Table19). In general, bicyclic-monoterpenes were more potent inducers ofmelanogenesis than monocyclic-monoterpenes, and within each of thesegroups, diols were more potent than alcohols, while non-hydroxylatedcompounds exhibited little or no activity (Table 19).

[0219] Bicyclic Monoterpenes

[0220] 1R,2R,3S,5R)-(−)-pinanediol was only slightly more potent than(1S,2S,3R,5S)-(+)-pinanediol (Table 19), indicating that melanogenicactivity of 2,3-cis/exo-pinanediol is relatively independent ofenantomeric configuration.

[0221] (1R)-(−)-trans-pinane-1,10-diol which contains a 2-hydroxymethylgroup, exhibited melanogenic potency almost identical to that of(1R,2R,3S,5R)-(−)-pinanediol (Table 19). These results indicate thatmarkedly different pinanediol structures may possess significantmelanogenic activity. Therefore, all pinanediol compounds, includingmethanol and dimethanol substituted pinanediol derivatives are claimedin this invention.

[0222] (1S,2S,5S,)-2-hydroxy-3-pinanone was about half as potent as(1R,2R,3S,5R)-(−)-pinanediol (Table 19), indicating that substitution ofa keto group for a hydroxyl group only partially reduces melanogenicactivity. Given this finding, and the fact that keto groups may readilybe converted to hydroxyl groups by chemical or biological processes, itis contemplated that substitution of a keto group for a hydroxyl groupin any of the compounds in this invention may result in retention ofmelanogenic activity. Therefore, all such keto-substituted compounds areclaimed in this invention.

[0223] (−)-Isopinochampheol, an alcohol closely related to(1R,2R,3S,5R)-(−)-pinanediol (also known as[−]-2-hydroxyisopinocampheol), possessed considerably less melanogenicactivity (Table 19). In addition, (−)-isopinochampheol resulted indetachment of cells from culture dishes at concentrations where(1R,2R,3S,5R)-(−)-pinanediol was a highly efficacious inducer ofmelanogenesis, indicating that the alcohol was more toxic than the diol.Similar results were obtained for (S)-cis-verbenol, another closelyrelated bicyclic alcohol, and (1R)-(−)-myrtenol, a pinene derivativewhich contains a methanol substituent group (Table 19). Therefore,results for pinanediol derivatives indicate that alcohols are lesspotent inducers of melanogenesis than diols when tested in S91 cells, inagreement with previous results for norbornane derivatives (Example 6,FIG. 2).

[0224] However, as noted previously, although 2-norbornanemethanolexhibited considerably less melanogenic activity than5-norbornene-2,2-dimethanol in S91 cells (Example 6, FIG. 2), itexhibited nearly equivalent cellular melanogenic activity in normalhuman epidermal melanocytes (Example 7, Table 12). Therefore, it iscontemplated that similar findings may be incurred by(−)-isopinochampheol, (S)-cis-verbenol, (1R)-(−)-myrtenol, and relatedalcohols when tested in normal human epidermal melanocytes. Moreover,all alcohol derivatives of the compounds of this invention are eithershown or contemplated to possess various degrees of melanogenicactivity, and are therefore claimed in this invention. Nonsubstitutedpinane enantiomers exhibited little or no melanogenic activity.

[0225] A mixture of 2,3-cis/exo- and 2,3-trans-bornanediol was found toexhibit approximately twice as much melanogenic activity as(1R,2R,3S,5R)-(−)-pinanediol (Table 19). Examination of purifiedstereoisomers indicated 2,3-cis/exo-bornanediol was more than twice aspotent as 2,3-trans-bornanediol (Table 19). Borneol, an alcoholderivative, possessed much less activity (Table 19). Results for thesebornane derivatives combined with those for the norbornane derivativesand pinane derivatives provide evidence that any bicyclic or multicycliccompound may provide a suitable framework for incorporation ofsubstituent groups that induce melanogenesis. Therefore, all suchcompounds are claimed in this invention.

[0226] Within the bicyclic compounds that were examined, pinane andbornane derivatives (Table 19) were more potent inducers ofmelanogenesis than norbornane derivatives (Example 6, FIG. 2; Example11, Tables 15 and 16). Unlike bicyclic norbornanes which contain nomethyl substituents, the bicyclic-monoterpene pinanes and bornanescontain three methyl substituents. Thus, it is contemplated that a rangeof substituents including but not limited to methyl groups may increasemelanogenic activity of bicyclic compounds.

[0227] Monocyclic Monoterpenes

[0228] Cis-p-menthane-3,8-diol and trans-p-menthane-3,8-diol were themost potent monocyclic monoterpenes examined (Table 19). However, thesepossessed much less melanogenic activity than any of thebicyclic-monoterpene diols examined (Table 19). Similar to results forthe bicyclic-monoterpenes, the alcohols exhibited only low levels ofmelanogenic activity, and were toxic at the higher concentrations tested(Table 19). Moreover, R-(+)-limonene, a non-hydroxylatedmonocyclic-monterpene exhibited little or no melanogenic activity.

[0229] Similar to the monocyclic monoterpene alcohols,trans-p-menthane-2,8-diol exhibited much less melanogenic activity thancis-p-menthane-3,8-diol or trans-p-menthane-3,8-diol. However, unlikethe alcohols, trans-p-menthane-2,8-diol was not toxic at the higherconcentrations tested (Table 19). Thus, based on results for bothbicyclic-monocyclic-monoterpenes, it is expected that diols will bepreferable to alcohols as melanogenic agents, not only because they aremore potent, but also because they appear to be less toxic.

[0230] Similar to cyclohexanediol, cis-p-menthane-3,8-diol andtrans-p-menthane-3,8-diol possess six member rings. However,cis-p-menthane-3,8-diol and trans-p-menthane-3,8-diol are markedly morepotent than either monocyclic hexanediol or pentanediol (Example 3 andTable 5). Thus, similar to bicyclic compounds, it is contemplated that arange of substituents including but not limited to methyl groups mayincrease melanogenic activity of monocyclic compounds. Enhancement ofmelanogenic potency of aliphatic diols by incorporation of methylsubstituents has been demonstrated previously (e.g., compare2,3-butanediol and 2,3-dimethyl-2,3-butanediol in Example 3, Table 5).TABLE 19 Fold Induction of Tyrosinase Relative to Controls 0.5 mM 1 mM2.5 mM 5 mM Bicyclic Monoterpenes (1R,2R,3S,5R)-(−)- 5.1× 17.6×   119×50.6× pinanediol (1S,2S,3R,5S)-(+)-  ND¹ 9.9× 68.4× 39.9× pinanediol(1R)-(−)-trans-pinane ND 14.3×  96.0× 36.8× 1,10-diol(1S,2S,5S,)-2-hydroxy- ND 4.1× 37.1× 94.9× 3-pinanone(−)-isopinocamnpheol 2.3× 3.3×  NA² NA (S)-cis-verbenol 1.0× 3.0× 15.5×NA (1R)-(−)-myrtenol³ 13.6×  17.6×   2.2× NA (1R)-(+)-α-pinane 1.2× 0.8× 0.9× NA (1S)-(−)-α-pinane 1.4× 1.1×  1.4×  1.2× 2,3-cis & trans- 9.9×37.3×  99.7× ND bornanediol 2,3-cis/exo-bornanediol 10.2×  28.5×   101×ND 2,3-trans-bornanediol 4.7× 12.8×  22.1× ND borneol 3.7× 2.1×  3.7× NDMonocyclic Monoterpenes cis-p-menthane-3,8-diol 1.6× 3.0× 17.1× 11.6×trans-p-menthane-3,8-diol 5.2× 13.2×  21.0×  7.3× sobrerol⁴ 1.8× 2.0× 2.3×  3.5× (−)-a-terpineol⁵ 6.2× 7.1×  4.7× NA (1R,2S,5R)-(−)-menthol⁶2.0× 1.3× NA NA (1S,2R,5S)-(+)-menthol⁷ 0.7× 1.3× NA NA R-(+)-limonene⁸1.2× 1.8×  1.6×  1.4×

What is claimed is:
 1. A composition for increasing the melanin contentof mammalian melanocytes comprising: a) an effective amount of one ormore compounds selected from the group consisting of: (i)bicyclic-monoterpene diols, (ii) pharmaceutically acceptable salts of(i), and (iii) prodrugs of (i); and b) a suitable carrier.
 2. A methodfor increasing the melanin content of mammalian melanocytes comprisingadministering to said melanocytes an effective amount of the compositionof claim
 1. 3. A composition for treating a skin proliferative disorderor a disorder of keratinization comprising: a) an effective amount ofone or more compounds selected from the group consisting of: (i)bicyclic-monoterpene diols, (ii) pharmaceutically acceptable salts of(i), and (iii) prodrugs of (i); and b) a suitable carrier.
 4. A methodfor treating a skin proliferative disorder or a disorder ofkeratinization in a mammal comprising administering to a mammal in needof such treatment an effective amount of the composition of claim
 3. 5.A composition for preventing a skin proliferative disorder or a disorderof keratinization comprising: a) an effective amount of one or morecompounds selected from the group consisting of: (i)bicyclic-monoterpene diols, (ii) pharmaceutically acceptable salts of(i), and (iii) prodrugs of (i); and b) a suitable carrier.
 6. A methodfor preventing a skin proliferative disorder or a disorder ofkeratinization in a mammal comprising administering to a mammal in needof such preventive treatment an effective amount of the composition ofclaim
 5. 7. A composition for altering or restoring pigmentation inmammalian skin, hair, wool or fur comprising: a) an effective amount ofone or more compounds selected from the group consisting of: (i)bicyclic-monoterpene diols, (ii) pharmaceutically acceptable salts of(i), and (iii) prodrugs of (i); and b) a suitable carrier.
 8. A methodfor altering or restoring pigmentation in mammalian skin, hair, wool orfur comprising administering to a mammal in need of such alteration orrestoration an effective amount of the composition of claim
 7. 9. Acomposition for treating a disease mediated by perturbations of theNO/cGMP/PKG pathway comprising: a) an effective amount of one or morecompounds selected from the group consisting of: (i)bicyclic-monoterpene diols, (ii) pharmaceutically acceptable salts of(i), and (iii) prodrugs of (i); and b) a suitable carrier; wherein saideffective amount is effective to directly stimulate NO synthesis withincells.
 10. A method for treating a disease mediated by perturbations ofthe NO/cGMP/PKG pathway in a mammal comprising administering to a mammalin need of such treatment an effective amount of the composition ofclaim
 9. 11. A composition for treating a disease mediated byperturbations of the NO/cGMP/PKG pathway comprising: a) an effectiveamount of one or more compounds selected from the group consisting of:(i) saturated C₇ to C₅₀ diols having the following structure:

 wherein each R is independently selected from R₁; R₂; hydroxyl, methyl,hydroxymethyl, —(CH₂)_(n)CH₃, —(CH₂)_(n)OH, —(CH₂)_(n)OR₁,—(CH₂)_(n)—CH(OH)—CHOH, —(CH₂)_(n)—CH(OH)—CH(OH)R₁,—(CH₂)_(n)—CH(OH)—(CH₂)—CH₂(OH), —(CH₂)_(n)—CH(OH)—(CH₂)_(n)—CH(OH)R₁ or—CH₂OR₁, wherein each n is independently an integer from 0-25; each R₁is independently selected from hydrogen; halogen; an acyl or amino acylgroup containing from one atom to twenty atoms, at least one of which iscarbon, nitrogen, oxygen, or sulfur; or a group containing from one atomto twenty atoms, one of which is carbon, nitrogen, oxygen, or sulfur,and R₂ is a linear, branched or unbranched, cyclic, bicyclic orpolycyclic group containing from one atom to fifty atoms, at least oneof which is carbon, nitrogen, oxygen, or sulfur; (ii) unsaturated C₇ toC₅₀ diols having the above structure; (iii) pharmaceutically acceptablesalts of (i); (iv) prodrugs of (i); (v) pharmaceutically acceptablesalts of (ii); and (vi) prodrugs of (ii); and b) a suitable carrier. 12.The composition of claim 11, wherein the C₇ to C₅₀ diol is selected fromthe group consisting of: (a) 5-norbornene-2,2-dimethanol, (b)norbornane-2,2-dimethanol, (c) 2,3-norbornanediol (exo or endo or cis ortrans), (d) 2,3-cis-exo-norbornanediol, (e)2-(propyl-1,2-diol)-norbornane, (f) 2,7-norbornanediol, (g)2-hydroxy-2-norbornanemethanol, (h)1-(exo-2-norbornyl-)-propan-1,2-diol, (i)1-(endo-2-norbornyl-)-propan-1,2-diol, (j)methyl-5-norbornene-2,3-dimethanol, (k)2,3-cis/exo-pinanediol([1R,2R,3S,5R]-[−]-pinanediol and[1S,2S,3R,5S]-[+]-pinanediol]), (l) (1R)-(−)-trans-pinane-1,10-diol, (m)2,3-cis/exo-bornanediol, (n) 2,3-trans-bornanediol, (o) camphanediol,(p) camphenediol, and (q) 2,3-trans-pinanediol.
 13. A method fortreating a disease mediated by perturbations of the NO/cGMP/PKG pathwayin a mammal comprising administering to a mammal in need of suchtreatment an effective amount of one or more compounds selected from thegroup consisting of: (i) saturated C₇ to C₅₀ diols having the followingstructure:

 wherein each R is independently selected from R₁; R₂; hydroxyl, methyl,hydroxymethyl, —(CH₂)_(n)CH₃, —(CH₂)_(n)OH, —(CH₂)_(n)OR₁,—(CH₂)_(n)—CH(OH)—CHOH, —(CH₂)_(n)—CH(OH)—CH(OH)R₁,—(CH₂)_(n)—CH(OH)—(CH₂)_(n)—CH₂(OH),—(CH₂)_(n)—CH(OH)—(CH₂)_(n)—CH(OH)R₁ or —CH₂OR₁, wherein each n isindependently an integer from 0-25; each R₁ is independently selectedfrom hydrogen; halogen; an acyl or amino acyl group containing from oneatom to twenty atoms, at least one of which is carbon, nitrogen, oxygen,or sulfur; or a group containing from one atom to twenty atoms, one ofwhich is carbon, nitrogen, oxygen, or sulfur, and R₂ is a linear,branched or unbranched, cyclic, bicyclic or polycyclic group containingfrom one atom to fifty atoms, at least one of which is carbon, nitrogen,oxygen, or sulfur; or (ii) unsaturated C₇ to C₅₀ diols having the abovestructure; (iii) pharmaceutically acceptable salts of (i); (iv) prodrugsof (i); (v) pharmaceutically acceptable salts of (ii); and (vi) prodrugsof (ii).
 14. The method of claim 13 wherein the C₇ to C₅₀ diol isselected from the group consisting of: (a) 5-norbornene-2,2-dimethanol,(b) norbornane-2,2-dimethanol, (c) 2,3-norbornanediol (exo or endo orcis or trans), (d) 2,3-cis-exo-norbornanediol, (e)2-(propyl-1,2-diol)-norbornane, (f) 2,7-norbornanediol, (g)2-hydroxy-2-norbornanemethanol, (h)1-(exo-2-norbornyl-)-propan-1,2-diol, (i)1-(endo-2-norbornyl-)-propan-1,2-diol, (j)methyl-5-norbornene-2,3-dimethanol, (k)2,3-cis/exo-pinanediol([1R,2R,3S,5R]-[−]-pinanediol and[1S,2S,3R,5S]-[+]-pinanediol]), (l) (1R)-(−)-trans-pinane-1,10-diol, (m)2,3-cis/exo-bornanediol, (n) 2,3-trans-bornanediol, (o) camphanediol,(p) camphenediol, and (q) 2,3-trans-pinanediol.